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Protocols References Resources Orders Biobricks


Week 3

Tuesday 17th July 2007

  1. Restriction Digests of transformed BioBricks (see Protocol 7):
Label BioBrick Plasmid Description Enzymes Expected Sizes
3/19A BBa_J23119 pSB1A2 strong constitutive promoter NheI, PvuI 680bp, 1430bp
1/9G BBa_R0062 pBB1A2 HSL and luxR EcoRI, PvuI 1460bp, 660bp
1/16P BBa_J04500 pSB1AK3 IPTG inducer and RBS PvuII, PvuI 2250bp, 1030bp, 730bp
4/11C BBa_p1010 pSB3K3 death gene BamHI, XhoI 190bp, 2390bp,840bp
1/5H BBa_E0040 pSB1A2 GFP no promoter Hime II, PvuI 1630bp, 1170bp
4/6D BBa_I52001 p5B4K5 High copy death gene AvaI, PvuI 920bp, 1470bp, 2120bp

Wednesday 18th July 2007

  1. Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
  2. Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
  3. Maia is extracting DNA from 3 samples of Pseudomonas Aeruginosa, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Protocol 8); only change was to shorten "shaking" time from 10 to 2 minutes.
  4. PCR trial run using Reddymix and Touch 2 done (see Protocol 9 for PCR).
Template Primers

XylR_prefix + XylR_suffix
Pr_prefix + Pr_suffix
Pr_prefix + XylR_suffix
Pu_prefix_EM + Pu_suffix_EM

pQF52 DntR_prefix + DntR_suffix
DmpR WT/24 DntR_prefix + DntR_suffix

Thursday 19th July 2007

  1. Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (see Protocol 9) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
    • XylR prefix and XylR suffix
    • Pr prefix and Pr suffix
    • Pr prefix and XylR suffix
    • Pu prefix and Pu suffix
    • DntR prefix and DntR suffix 2

Friday 20th July 2007


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